Methods to increase the photostability of dyes

Abstract

The presently disclosed subject matter provides dyes having an improved photostability, biosensors comprising such dyes, and methods of use thereof, including methods for detecting target molecules in a sample under test and for live-cell imaging. The dyes can include a binding member, including a biomolecule or fragments thereof, which can interact with target molecules of interest and can be specific to a given conformational state or covalent modification, e.g., phosphorylation, of the target molecule. The presently disclosed dyes can be used for detecting changes in the binding, conformational change, or posttranslational modification of the target molecule.

That which is claimed: 
     
       1. A dye of Formula (II); 
       
         
           
           
               
               
           
         
         wherein:
 m is an integer from 1 to 4; 
 X 2  is halogen; 
 R 1 , R 2 , R 3 , R 6 , and R 7  are each independently selected from the group consisting of H, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, hydroxyalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aminoalkyl, acyloxyl, alkylaminoalkyl, alkoxycarbonyl, alkyleneaminoalkyl, alkylenesulfate, —(CH 2 ) p —SO 3 , —(CH 2 ) p —N + (CH 3 ) 2 —(CH 2 ) p NCS, and —(CH 2 ) p —NH—CO—CH 2 —X 2 ; wherein X 2  is halogen; 
 R 5  is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, hydroxyalkyl, hydroxycycloalkyl, alkoxycycloalkyl, aminoalkyl, acyloxyl, alkylaminoalkyl, alkoxycarbonyl, alkyleneaminoalkyl, alkylenesulfate, —(CH 2 ) p —SO 3 , —(CH 2 ) p —N + CH 3 ) 2 —(CH 2 ) p —NCS, and —(CH 2 ) p —NH—CO—CH 2 —X 2 ; wherein X 2  is halogen; 
 R 8  is selected from the group consisting of H, alkyl, substituted alkyl, hydroxyl, amino, nitro, alkoxyl, and hydroxyalkyl; 
 Y 1  is H; 
 Y 2  is selected from the group consisting of H, —NH 2 , —SO 3   − , —(CH 2 ) t OH, —(CH 2 ) t NH 3   + X 2   − ; CH 3 CONH—, X 2 CH 2 CONH—, HO(CH 2 ) 2 —S—CH 2 CONH—, SuOCOCH 2 OCH 2 CON(CH 3 )—, —CH 2 —NH—C(═O)—O—(CH 3 ) 3 , —(CH 2 ) t —NH 2   + —(CH 2 ) s —SO 3   − , —(CH 2 ) t —NH—C(═O)—(CH 2 ) s —X 2 , a succinimidyl ester, a hydroxysuccinimide, a maleimide, a haloacetyl, a pyridyl disulfide, a hydrazine, an ethyldiethylamino propylcarbodiimide, 
 
       
       
         
           
           
               
               
           
         
         
            or a protecting group, including t-butoxycarbonyl and carbobenzoxy; 
           and 
           wherein each p, r, s and t is independently an integer from 1 to 8; and Su is succinimidyl ester; 
           and salts thereof. 
         
       
     
     
       2. The dye of  claim 1 , wherein Y 1  is selected from the group consisting of: —(CH 2 ) 2 OH, —CH 2 —NH 3   + X 2   − ; —CH 2 —NH 2   + —(CH 2 ) 3 —SO 3   − ; —CH 2 —NH—C(═O)—CH 2 —X 2 ; —CH 2 —NH—C(═O)—O—(CH 3 ) 3 ; 
       
         
           
           
               
               
           
         
         wherein X 2  is halogen selected from the group consisting of Cl and I. 
       
     
     
       3. A dye selected from 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         and salts thereof.